While the polymerase chain reaction (PCR) and related techniques are highly useful for a variety of applications, the amplification of non-target nucleic acids due to undesired side-reactions can present a significant problem. Such side reactions can occur as a result of mis-priming of non-target nucleic acids and/or primer oligomerization, sometimes referred to as primer-dimer formation, and the subsequent amplification of these priming artifacts. This is especially true in applications in which PCR is carried out using a mixture of nucleic acids with significant background nucleic acids while the target nucleic acid is present in low copy number (see, e.g., Chou et al., Nucl. Acids Res. 20:1717 (1992)). The generation of non-specifically amplified products has been attributed at least in part to DNA polymerase activity at ambient temperature that extends non-specifically annealed primers (see, e.g., Chou et al., supra; Li et al., Proc. Natl. Acad. Sci. USA 87:4580 (1990)). Accordingly, inhibition of DNA polymerase activity at ambient temperature is beneficial in controlling the generation of secondary amplicons.
Several “hot start” techniques have been described which reportedly decrease the formation of undesired secondary amplification products. According to certain “manual hot start” techniques, a component critical to DNA polymerase activity (e.g., divalent ions and/or the DNA polymerase itself) is not added to the reaction mixture until the temperature of the mixture is high enough to prevent non-specific primer annealing (see, e.g., Chou et al., supra; D'Aquila et al., Nucl. Acids Res. 19:3749 (1991)). Less labor-intensive techniques employ the physical separation or reversible inactivation of at least one component of the amplification reaction. For example, the magnesium or the DNA polymerase can be sequestered in a wax bead, which melts as the reaction temperature increases, releasing the sequestered component only at the elevated temperature. According to other techniques, the DNA polymerase is reversibly inactivated or modified, for example by a reversible chemical modification of the DNA polymerase, the binding of an antibody to the DNA polymerase, or oligonucleotide molecules that bind to the DNA polymerase (see, e.g., U.S. Pat. Nos. 5,677,152 and 5,338,671; and Dang et al., J. Mol. Biol. 264:268 (1996)). At an elevated reaction temperature, the chemical modification is reversed, or the antibody molecule or oligonucleotide molecule is denatured, releasing a functional DNA polymerase. However, some of these techniques appear to be less than optimal, in that some DNA polymerase activity is detectable at lower reaction temperatures despite the inactivation, or they require extended exposure of the reaction mixture at high temperatures to fully activate the DNA polymerase, which may result in permanent inactivation of some components of the reaction mixture.
Certain currently used nucleic acid amplification techniques include a step for detecting and/or quantifying amplification products that comprise a nucleic acid dye, for example, but not limited to, SYBR® Green I (Life Technologies, Carlsbad, Calif.), including certain real-time and/or end-point detection techniques (see, e.g., Ririe et al., Anal. Biochem. 245:154 (1997)). Typically the nucleic acid dye associates with double-stranded segments of the amplification products and/or primer-template duplexes and emits a detectable fluorescent signal at a wavelength that is characteristic of the particular nucleic acid dye. Certain amplification methods comprise a detection step for evaluating the purity of the amplification product(s) associated with the nucleic acid dye, for example but not limited to, post-PCR dissociation curve analysis, also known as melting curve analysis. Since the melting curve of an amplicon is dependent on its length and sequence (among other things), amplicons can generally be distinguished by their melting curves (see, e.g., Zhang et al., Hepatology 36:723 (2002)). A dissociation or melting curve can be obtained during certain amplification reactions by monitoring the nucleic acid dye fluorescence as the reaction temperatures pass through the melting temperature of the amplicon(s). The dissociation of a double-stranded amplicon is observed as a sudden decrease in fluorescence at the emission wavelength characteristic of the nucleic acid dye. According to certain dissociation curve analysis techniques, an amplification product is classified as “pure” when the melting curve shows a single, consistent melting temperature, sometimes graphically displayed as a peak on a plot of the negative derivative of fluorescent intensity versus temperature (−dF/dt vs. T). In contrast, the appearance of multiple peaks in such a dissociation curve from a single-plex amplification typically indicates the presence of undesired side reaction products. When such nucleic acid dye-based amplification product detection techniques are employed, it is often desirable to: 1) at least decrease and preferably eliminate the formation of undesired side-reaction products, and 2) at least decrease and preferably eliminate fluorescence peaks resulting from the denaturing of double-stranded segments of other nucleic acids, i.e., non-amplification products (e.g., primer dimers) and/or non-specific amplification products (e.g., due to mis-priming events).
Certain other amplification techniques may also yield undesired amplification products due to, among other things, non-specific annealing of primers, ligation probes, cleavage probes, promoter-primers, and so forth, and subsequent enzyme activity at sub-optimal temperatures. For example, while reaction components are being combined, often at room temperature, or while the reaction composition is being heated to a desired reaction temperature. At least some of these techniques can benefit from a reduction in background fluorescence. Thus, there is a need for compositions and methods that decrease and/or eliminate 1) the formation of undesired side-reaction products and 2) the background fluorescence resulting from these undesired side-reaction products.